The aim of the proposed research is to elucidate the quaternary structure and mechanism of action of the enzyme hydrogenase. Pure hydrogenase from Chromatium, Proteus vulgaris, and Escherichia coli will be studied to establish the structure of the active iron-sulfur site, the nature of its reaction with substrate and inhibitors, and the mechanism by which the enzyme cleaves hydrogen and forms an enzyme hydride intermediate. The different forms of the enzyme (reduced, oxidized, oxidized-oxygenated) will be identified and differentiated by activity, optical and EPR spectra, and will establish the number of electrons accepted by the enzyme from the substrate. The same techniques will be applied to the interaction of the enzyme with the inhibitors oxygen, carbon monoxide, and nitric oxide and will establish the state of the enzyme they react with and the chemical basis of the inhibition. Experiments will be carried out to demonstrate conclusively the formation of an enzyme hydride intermediate. This involves transfer of the hydride from the enzyme directly to a suitable substrate where the hydrogen would be bound to carbon in a non-exchangeable form. This research will also investigate the regulation of hydrogenase synthesis in microorganisms which will establish the role of the enzyme in microbial physiology, define conditions for induced synthesis of the enzyme at high levels, and permit the mapping of the hydrogenase gene and its transfer to organisms lacking the enzyme. Approaches are outlined for the isolation of mutant forms of hydrogenase with altered properties. Of particular interest is the isolation of an oxygen insensitive enzyme which retains full catalytic activity in the presence of oxygen.